1. Field of the Invention
The present invention relates to a measuring apparatus for executing, for example, fluorescence correlation spectroscopy (FCS) in which light is applied to a sample, and fluorescence emitted from a fluorescent material contained as an indicator in the sample, and more particularly, to a measuring apparatus with an alignment detection unit for focusing light on the sample.
2. Description of the Related Art
The basic technique of the present invention relates to FCS that utilizes a confocal laser scanning microscope. In FCS, protein or colloidal particles labeled by a fluorescent material are floated in a solution within the viewing field of a confocal optical microscope, and a laser beam is applied to the fluorescent material to excite it, thereby analyzing variations in the intensity of fluorescence based on the Brownian motion of the fine particles to acquire a auto-correlation function and detect the number of target fine particles or translational diffusion speed of them.
For instance, Jpn. PCT National Publication No. 11-502608 discloses a method and apparatus in which a laser beam is applied by confocal laser scanning microscope to a sample labeled with a fluorescent material and placed on a sample stage, thereby analyzing variations in the intensity of fluorescence emitted from the sample, and detecting, for example, a statistical property, such as a translational diffusion coefficient, of fluorescent molecules, or the interaction of the molecules.
Further, U.S. Pat. No. 6,071,748 discloses an apparatus for condensing a laser beam onto a sample that contains a fluorescent material, and measuring the intensity or life of fluorescence emitted therefrom.
In methods for applying a laser beam to a sample to excite a fluorescent material contained therein, and analyzing fluctuation in the intensity of fluorescence emitted from the sample, a sample container called a microplate is often used. If a microplate is used, a large number of samples can be simultaneously received in a plurality of wells formed in the plate, and be measured individually. Further, since an extremely small amount of sample material of a microliter order can be measured, it is not necessary to prepare a large amount of sample material.
In fluorescence correlated spectroscopy, the bottom of a microplate is formed of, for example, glass, and an objective lens is located below the microplate. The light from a light source is applied to a sample contained in each well of the microplate through the bottom thereof, thereby detecting the behavior of each molecule of the sample. When a microplate with a transparent bottom is used, an optical signal, such as a fluorescence signal, generated by the sample can be measured by an excitation optical system. Namely, a measuring device of a simple structure can be used, which is advantageous in designing the device.
Further, when a light beam is directly condensed onto a sample in each well through the bottom of the microplate, it is necessary to accurately adjust the focal point of the beam to reliably guide it to the sample. However, since an objective lens of high magnification is used as a condensing lens, the focal depth is extremely shallow. In this condition, to accurately adjust the focal point, a skilled operator is needed.
To solve this problem, the following techniques, for example, have been proposed.
Jpn. PCT National Publication No. 2002-541430 discloses a technique of detecting the position of the bottom of a microplate simply using a measuring optical system. Namely, the light from a light source is condensed onto a sample through the microplate bottom, and the focal point of the incident light is gradually moved along the optical axis, thereby measuring the intensity of the light directly reflected from the microplate bottom. The position on the optical axis, at which the intensity peak value is detected, is determined to be the bottom of the microplate, and the focal point of light in each well of the microplate is set based on the determination result.
Jpn. PCT National Publication No. 2002-542480 discloses a technique of receiving, by a CCD camera, the beam emitted from a light source, detecting the position of the beam spot on the light-receiving surface of the camera to determine, for example, the bottom position of the plate, and adjusting the focal point of the light source in accordance with the detected bottom position.
Jpn. Pat. Appln. KOKOKU Publication No. 02-59963 discloses a technique of detecting a deviation in the focal point of a sample, based on a deviation in the point, on the light-receiving surface of a differential diode, of the light emitted from a light source, thereby moving a sample stage along the optical axis, based on the detected deviation, in order to converge the light spot of the light source on the surface of the sample.